Capillary electrophoresis has achieved a remarkably rapid development from its introduction in the early 1980s. This technique miniaturizes the electrophoretic process and presents significant advantages over traditional slab gel electrophoretic techniques. While fused silica capillaries are the most frequently used separation format, the method was also transferred to microchips (Belder, D., and Ludwig, M. Surface modification in microchip electrophoresis. Electrophoresis. 2003; 24, 3595-3606, Becker, H., Locascio, L. E., Polymer microfluidic devices. Talanta 2002, 56, 267-287.) Most of materials used to prepare separation channels or capillaries for capillary electrophoresis (CE) contain ionizable groups on their surface that are responsible for so-called electrokinetic potential or ζ-potential. This potential is a cause of electroosmotic flow (EOF). The presence of EOF and especially its uneven distribution along the electrophoretic capillary or channel causes disturbances called eddy migration and loss of resolution during electrophoretic separation. To suppress EOF, a good wall coating eliminates ζ-potential at the wall and/or increases viscosity inside the electric double layer. To reduce ζ-potential, the coating may react with charged groups incorporated in the wall (silanol groups in the case of fused silica capillary). To some extent, compounds with an opposite charge than the ionizable groups of the wall can be also used to titrate ζ-potential.
A number of various wall coatings have been proposed and developed to eliminate EOF and adsorption of analytes in fused silica capillaries. A vast majority of them merely reduced EOF and did not eliminate it completely. Frequently a dynamic wall coating was formed by simply adding an active ingredient to the background electrolyte. It adsorbed on the wall and reduced capillary surface charge and/or viscosity of solution in the electric double layer. Dynamic wall coatings are popular because of the simplicity of their preparation. However, they do not eliminate electroosmotic flow completely. Among many dynamic coatings, a dynamic coating based on guaran has been developed (Liu, Q., Lin, F., Hartwick, R. A. Capillary zone electrophoretic separation of basic proteins and drugs using guaran as a buffer modifier. Chromatographia 1998, 47, 219-224).
To eliminate EOF completely, static wall coatings have to be applied. Typically, a static wall coating is made of two layers: an intermediate layer and a hydrophilic polymer layer. A bifunctional reagent that reacts with both the capillary surface and functional groups of the polymer molecule usually forms the intermediate layer. The first polymer used for the preparation of a static wall coating was a linear polyacrylamide attached to the fused silica capillary wall by γ-methacryloxypropyltrimethoxysilane (Hjertén, S., Coating for electrophoresis tube. U.S. Pat. No. 4,680,201). More hydrolytically stable coating was obtained when polyacrylamide was attached to the silica wall by using a Grignard reagent with an olefinic moiety, e.g., vinylmagnesium bromide after activating silanol groups by a reaction with thionyl chloride (Novotny, M. V.; Cobb, K. A., and Dolnik, V., Suppression of electroosmosis with hydrolytically stable coatings. U.S. Pat. No. 5,074,982; Novotny, M. V.; Cobb, K. A., and Dolnik, V., Suppression of electroosmosis with hydrolytically stable coatings. U.S. Pat. No. 5,143,753). Polyacrylamide is, however, hydrolytically unstable at high pH and hydrolyzes forming poly(acrylic acid). The presence of carboxylic groups leads to generation of ζ-potential on the wall and to an increase of EOF. A more stable wall coating is usually obtained if acrylamide is replaced with its derivative having some substituents on nitrogen (Dolnik, V. and Chiari, M., Compounds for molecular separations. U.S. Pat. No. 6,074,542).
Thermal immobilization of a polymer on a capillary wall is another way how to anchor a polymer on the capillary wall. Schomburg and coworkers proposed a poly(vinyl alcohol) (PVA) coating fixed thermally to the wall by heating the capillary at 140.degree.C. They assumed that formation of a permanent PVA coating was based on PVA insolubility in water after the thermal treatment and expected PVA to form semi-crystalline highly associated structures, which were not covalently bound to the fused silica capillary. PVA molecules became more strongly associated by hydrogen bridges and water molecules could not penetrate microcrystalline domains. The authors expressed their opinion that this was a unique property of PVA. In the pH range of 5-9, the PVA coating did not, however, completely eliminate EOF and the coated capillaries exhibited a pH-independent electroosmotic mobility of 1.2×10−9 m2V−1s−1 as measured in 20 mM sodium phosphate (Schomburg, G. and Gilges, M., Deactivation of the inner surfaces of capillaries. U.S. Pat. No. 5,502,169). The procedure was further modified to make a PVA wall coating on a glass microchip. A newly introduced PVA coating crosslinked with glutaraldehyde should improve the stability of the coating. No heating is necessary, just drying is sufficient to provide a stable wall coating (Belder, D., Deege, A., Husmann, H., Kohler, F., and Ludwig, M. Cross-linked poly(vinyl alcohol) as permanent hydrophilic column coating for capillary electrophoresis. Electrophoresis. 2001; 22, 3813-3818). Thermal immobilization was also applied to hydroxyethyl cellulose (HEC) and hydroxypropyl cellulose (HPC) (Shen, Y. and Smith, R. D. High-resolution capillary isoelectric focusing of proteins using highly hydrophilic-substituted cellulose-coated capillaries. J. Microcol. Sep. 2000; 12, 135-141). The authors found that the wall coating was stable if the silica capillaries were heated at 140° C. for 20 min rather than just being dried at room temperature for 4 days. From this observation they concluded that a chemical reaction must have occurred between cellulose derivatives and fused silica capillary inner wall.
There are several types of galactomannans, a class of linear polysaccharides with 1,4 linked β-D-mannopyranosyl units and 1,6-linked α-D-galactopyranosyl side groups (Dolník, V., Gurske, W. A. and Padua, A.: Galactomannans as a sieving matrix in capillary electrophoresis. Electrophoresis 2001, 22, 707-719). The four most important galactomannans are locust bean gum, tara gum, guar gum (guaran), and fenugreek gum, which differ by the frequency of galactosyl side group attachment to the polymannose. The ratio of D-mannosyl to D-galactosyl units is approximately 3.8:1 for locust bean gum, 3:1 for tara gum, 1.8:1 for guar gum, and about 1:1 for fenugreek gum. Locust bean gum, guar gum, and tara gum are commercially produced and have various applications in the food industry and as an additive to fracturing fluids in the petroleum industry. Guar gum is obtained from the endosperm portion of the legume seed (Cyamopsis tetragonoloba) that grows mainly on the Indian subcontinent and in some parts of Texas and Oklahoma. Typical guar gum contains 75-85% of galactomannan, 8-14% water, 5-6% proteins, 2-3% fiber, and 0.5-1% ash. Guar gum shows an excellent resistance to shear degradation (Maier, H., Anderson, M., Karl, C., Magnus on, K., in: Whistler, R. L., BeMiller, J. N. (Eds.), Industrial Gums. Polysaccharides and Their Derivatives, Academic Press, San Diego 1993, pp. 181-226).
Solubility of polysaccharides and viscosity of their solutions significantly depends on branching of polysaccharide chains. Whereas cellulose is insoluble in water, as numerous hydrogen bridges between closely placed linear polysaccharide chains prevent its solubilization, highly branched polysaccharides, such as dextran, can be easily and quickly dissolved in water. When cellulose is derivatized with hydroxyalkyl groups, their presence keep polysaccharide chains apart and cellulose derivative becomes soluble in water (Whistler, R. L., BeMiller, J. N. (Eds.), Industrial Gums. Polysaccharides and Their Derivatives, Academic Press, San Diego 1993). Similarly, locust bean gum, where polymannose backbone fibers are kept apart only by 1 galactose side group per about 3.8 mannose units, is less soluble in water than, e.g., guar gum, where polymannose backbone fibers are kept apart by 1 galactose side group per about 1.8 mannose units (Maier, H., Anderson, M., Karl, C., Magnus on, K., in: Whistler, R. L., BeMiller, J. N. (Eds.), Industrial Gums. Polysaccharides and Their Derivatives, Academic Press, San Diego 1993, pp. 181-226). Solubility of polysaccharides can be reduced by enzymatic reaction, when side chains and side groups are hydrolytically cleaved from a polysaccharide backbone (Whistler R., Conversion of Guar Gum to Gel-Forming Polysaccharides by the Action of Alpha-Galactosidase. U.S. Pat. No. 4,332,894, 1982).